Many bacterial organisms modulate their response to environmental and metabolic cues with riboswitches. The discovery of these short cis-acting RNA elements has drastically changed our understanding of gene regulatory mechanisms in prokaryotes. Riboswitches are especially prevalent in Gram-positive bacteria, exemplified by Bacillus subtilis as a model organism, but are also found to control essential genes in important pathogens such as Bacillus anthracis, Staphylococcus, Enterococcus, Streptococcus, Listeria, Clostridium, and Mycobacterium. Due to their abundance in bacterial pathogens and their essential and specific nature, riboswitches are prime targets for drug and biotechnological development. Arguably the first riboswitch family identified, the T box riboswitch is unique from other characterized riboswitch families in that its effector is not a small molecule, but an essential macromolecule, tRNA. It is also a rare riboswitch system where ligand (tRNA) binding turns on essential gene expression rather than off. This makes the T box riboswitch an especially attractive antibiotic target because small molecules disrupting T box-tRNA interaction would trap the regulated gene in the off state, and the pathogens cannot easily acquire antibiotic resistance through loss-of-function riboswitch mutations. Mechanistically, the T box riboswitch is capable of two interesting functions: 1) recruiting a cognate tRNA from the intracellular tRNA pool through a process somewhat resembles the decoding process inside the ribosome, and 2) sensing the aminoacylation status of the bound tRNA to regulate genes involved in amino acid metabolism and tRNA aminoacylation. Standing in sharp contrast to the fast evolving field of metabolite-sensing riboswitches, mechanistic description of T boxes remain as low-resolution schematics in reviews, until last year when efforts from our group and others pushed the understanding of selective tRNA recognition to near atomic resolution. The combination of research tools including crystal structure determination, small angle X-ray scatting measurement, mutagenesis, chemical probing, and UV crosslinking analyses form the basis for the proposed investigations in this proposal. We aim to 1) provide a thorough understanding of the tRNA recruitment process by T box Stem I; 2) provide a holistic understanding of tRNA aminoacylation sensing by the Antiterminator domain of T box; and 3) understand the structure-function of an atypical class of T box riboswitch found in Mycobacterium tuberculosis, whose tRNA recognition mechanism cannot be explained by our current mechanistic model.